Artificial insemination in the emu (Dromaius novaehollandiae ...

Animal Science Papers and Reports vol. 22 (2004) no. 3, 315-323
Institute of Genetics and Animal Breeding, Jastrzębiec, Poland
Artificial insemination in the emu
(Dromaius novaehollandiae): effects of numbers
of spermatozoa and time of insemination
on the duration of the fertile period*
Ireneusz Artur Malecki**, Graeme Bruce Martin
School of Animal Biology (M085), Faculty of Natural and Agricultural Sciences
The University of Western Australia, Crawley, WA 6009, Australia
(Received July 15, 2004; accepted September 15, 2004)
In emus, the duration of the fertile period was measured following a single artificial insemination
(AI) and investigated the effect of time of AI in the egg cycle on the duration of the fertile period.
Semen was collected by artificial cloaca, pooled and used undiluted for AI within 30 minutes. For
insemination, a female was followed until she assumed the voluntary crouch. A speculum was then
inserted into the cloaca and an insemination straw introduced into the vagina to a depth of 1-2 cm, and
semen deposited. Following a single insemination with 100, 200 or 400 million spermatozoa, female
emus laid fertilized eggs for 10.0±0.4, 12.0±0.9, and 15.0±0.6 days. When 400 million spermatozoa
were used for insemination on Day 1, 2 or 3 of the oviposition cycle, the duration of the fertile period
appeared to change in a day-dependent manner. After AI on Day 1, female emus laid fertilized eggs
for 15.8±1.1 days, after AI on Day 2 for 12.5±2.2 days, and after AI on Day 3 for 10.0±1.5 days. The
results suggest that female emus need to be inseminated the day after oviposition to maximize the
duration of their fertile period.
KEY WORDS: artificial insemination / emu / fertility / oviposition / ratitae / sperm storage
The emu has adapted well to farming conditions, but monogamous mating does not
*Supported by the Rural Industries Research & Development Corporation in Australia
**Corresponding author: e-mail: imalecki@agric.uwa.edu.au
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I.A. Malecki and G.B. Martin
allow efficient production systems to develop and slows genetic improvement. Artificial
insemination (AI) is thus being developed for emu farming as an alternative to natural
mating. Naturally mated female emus store spermatozoa in the tubules in the oviduct
and release them over a period of time, termed the „fertile period” [Lake 1975, Birkhead
1988], during which up to 6 eggs can be fertilized [Malecki et al. 1998, Malecki and
Martin 2002b]. When female emus are inseminated artificially with doses equal to or
exceeding the average ejaculate volume, the fertile period only corresponds to the time
needed to lay five eggs. However, these are average values and the period during which
all females lay fertilized eggs could last for only nine days, effectively guaranteeing
only three fertilized eggs as emus lay eggs once every three days [Malecki and Martin
2002a]. This suggests that when breeding emus by AI, the number of eggs per AI may
be too low for the system to be viable. Since the duration for which females can store
sperm in their tubules is highly variable in emus [Malecki and Martin 2000a], as it is in
poultry [Bakst et al. 1994], selecting for longer duration of sperm storage could result
in longer insemination intervals thus increasing the number of fertilized eggs produced
per insemination. On the other hand, the minimum number of spermatozoa required
for AI could allow large number of females to be sired by a single male leading to a
substantial reduction in the costs of keeping males.
In the emu, the minimum numbers of spermatozoa that would guarantee maximum
fertility have not been investigated. However, it has been studied in domestic birds,
such as the chicken, in which 50 to 100 million spermatozoa need to be introduced
weekly [Taneja and Gove 1961, Wishart 1987] and the turkey, in which 100 to 200
million are needed to maintain the maximum flock fertility [Sexton 1977, Brillard and
Bakst 1990]. In the absence of large numbers of emus to test a range of sperm doses,
we approached the problem theoretically. The number of spermatozoa required in a
single insemination dose could be estimated from the storage capacity of the tubules,
termed the „sperm storage tubules” (SSTs), as their numbers and capacity determine
how many spermatozoa can be retained in the oviduct. Theoretically, there should be
sufficient spermatozoa in the tubules to fertilize an entire clutch [Birkhead and Moeller
1992]. From the relationship between the female body weight and the number of
SSTs [Birkhead and Moeller 1992], the utero-vaginal region of a 50 kg female emu
should contain about 27,000 tubules. Based on the dimensions of emu spermatozoa
and tubules [Malecki 1997], each tubule could hold about 400 spermatozoa and, if the
tubules were filled to capacity, the entire utero-vaginal junction would contain about
11 million spermatozoa. This situation is, however, unusual because tubules are rarely
filled to their maximum capacity and some contain no spermatozoa at all [Compton
and Van Krey 1979, McIntyre and Christensen 1983, Brillard et al. 1987, Brillard and
Bakst 1990, Briskie and Montgomerie 1993]. Nevertheless, if we assume that SSTs
are populated to their capacity and SST spermatozoa represent about 1-2% of those
deposited during insemination into the female’s vagina [Brillard and Bakst 1990, Brillard
1993], then female emus would require 100-200 million spermatozoa in an AI dose.
This dose should produce a fertile period corresponding to the clutch duration.
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The numbers of spermatozoa that will populate the tubules, and thus the duration
of the fertile period, can also be affected by the timing of insemination in the oviposition
cycle. In poultry, the transport of sperm to the SSTs appears to be affected by
oviducal contractions caused by oviposition and, the closer to oviposition AI is carried
out, the lower the fertility rate [Brillard et al. 1987]. As female emus lay eggs every
three days [Malecki and Martin 2002a], inseminations on Day 3 of the cycle (day of
oviposition) should result in shorter fertile period than those carried out on Day 1 or
2 of the cycle.
We therefore determined the number of spermatozoa required in an AI dose to
produce the maximum fertile period and the optimum time of insemination in relation
to day of oviposition. Our hypothesis was that, when female emus are inseminated with
100-200 million spermatozoa, their fertile period will correspond to the time of clutch
duration, and that the fertile period will shorten as the time of insemination approaches
the proceeding oviposition.
Material and methods
The experiments were approved by the Animal Experimentation Ethics Committee
of the University of Western Australia. We used adult emus (age 8 to 12 years) held at
the Shenton Park Field Station of the University of Western Australia (32°13’ S and
115°38’E). The birds were kept in pens that contained natural vegetation consisting of
grass, shrubs and trees, and they were provided ad libitum with water and a commercial
emu breeder ration. The females were initially penned individually adjacent to pens
that each contained a single male. Pen area was about 80 m 2 for females and about
40 m 2 for males. The study was carried out over two consecutive breeding seasons:
Experiment 1 in the first, and Experiment 2 in the second.
Experiment 1
To determine the number of spermatozoa required in an AI dose that would produce
maximum duration of the fertile period, 12 females (four per AI dose) were inseminated
once with fresh semen containing either 100, 200 or 400 million spermatozoa.
Inseminations were carried out between 09.00 and 10.00 a.m. on Day 1 of the egg
cycle (the day after oviposition), after female emus had laid two eggs, and eggs were
collected for the next four weeks.
Experiment 2
To determine the optimum time of AI that would guarantee maximum duration of
the fertile period, we used six females that were randomly assigned to three treatments:
Day 1 (the first day after oviposition, 15-17 hours after the last oviposition and 55-57
hours prior to the next oviposition), Day 2 (second day after oviposition, 31-33 hours
prior to the next oviposition) or Day 3 (third day after oviposition, 7-9 hours prior to
the next oviposition). Females were inseminated once with fresh semen containing 400
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I.A. Malecki and G.B. Martin
million spermatozoa between 09.00 and 10.00 a.m. Females were inseminated after two
eggs had been laid and eggs were collected until the end of the fertile period, defined
as when two consecutive unfertilized eggs were laid. We used a latin square design as
potentially every female could be tested for every AI time within one breeding season.
However, laying that season was not as good as anticipated and one female completed
only one treatment, four females completed two treatments and one completed three
treatments. As a result, each treatment had four replicates.
Semen collection and preparation
Semen was collected from four males using an artificial cloaca [Malecki et al. 1997],
taken to the laboratory and transferred by a graduated glass pipette from the collection
vial to the storage vial in order to separate semen from contaminant particles that
sometimes entered the artificial cloaca from the male’s feathers during the collection.
Spermatozoa concentration was then estimated with a Shimadzu spectrophotometer
and calculated from the standard curve. Ejaculate volume ranged from 0.4 to 1.2 mL
and each ejaculate contained 1,2-3,5 × 10 9 spermatozoa. The AI pool was made using
equal numbers of spermatozoa from each male. The concentration of spermatozoa in a
pool was subsequently confirmed with a spectrophotometer and the volume that would
contain the required number of spermatozoa was then estimated.
Artificial insemination
The female is followed until she voluntarily assumes the crouching position (Photo
1-A). Then the inseminator lowers himself behind the female and places his hands on her
back to provide stimulation, to which the female responds by bending her neck, raising
her tail and exposing the vent allowing access to the cloaca (Photo 1-B). Subsequently,
the inseminator kneels on the ground and puts his leg on the female’s back to continue
stimulation so the female remains crouched (Photo 1-C). If that was inadequate, instead
of the inseminator’s leg, an assistant would put his hands onto the female’s back and
press gently. The speculum fitted with lighting is then inserted into the cloaca so the
vaginal opening could be seen. The insemination straw, mounted on a tuberculin syringe,
is then introduced into the cloaca and inserted into the vagina until resistance is felt
(approximately 1-2 cm in depth). Deeper inseminations were not performed to avoid
irritation and possible cessation of laying (I. Malecki – personal observation). A dose
of sperm is then introduced into the vagina as the inseminating straw was gradually
withdrawn (Photo 1-D).
Data analysis
Eggs were stored for up to two weeks and then opened and fertilization status was
determined by the appearance of the germinal disc. Eggs were assumed fertilized when
the germinal disc contained a blastoderm, or not fertilized when a blastoderm was
absent [Malecki and Martin 2002b]. The duration of the fertile period was determined
as the number of days from day of insemination to the day the last fertilized egg was
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Artificial insemination in the emu
Photo 1. A – female emu begins to crouch voluntarily after being followed. B – crouching female emu
stimulated by rubbing her sides and back. Note the bending of the neck. C – one-person insemination of
the emu. While insemination is performed, the person maintains stimulation by pressing the emu’s back
with his leg. D – insemination of the emu using a speculum fitted with a light source and the insemination
straw mounted on a tuberculin syringe.
laid. One-way ANOVA was used to analyse data, with the SuperANOVA TM software
(Abacus Concepts, Inc., Berkeley, CA); data are presented as means ±SEM and P

I.A. Malecki and G.B. Martin
lasted for 15.8±1.1 days, after AI on Day 2 it lasted for 12.5±2.2 days and after AI on
Day 3 it lasted for 10.0±1.5 days.
Only the largest dose of spermatozoa produced a fertile period that corresponds to
the duration of the emu fertile period following artificial insemination and it exceeded
the previously estimated period of maximum flock fertility following artificial insemination
[Malecki and Martin 2002a]. Loss of spermatozoa viability during collection and
Figure 1. Effect of spermatozoa numbers (A) and time of artificial insemination (B) on the duration of the
fertile period in female emus. Bars with different letters differ significantly at P

Artificial insemination in the emu
tions of sperm storage. Moreover, an improvement could be sought by optimizing the
efficiency of the artificial insemination technique. Shallow inseminations can result in
a loss of spermatozoa from the vagina thus reducing the number of spermatozoa that
can populate the sperm storage tubules [Biellier et al. 1961, Ogasawara and Rooney
1965, Reinhart and Fiser 1983]. In the emu, unforced depth of insemination (no resistance
to the insemination straw) can vary from 1 to 6 cm between and within females (I.
Malecki – personal observation) meaning that the inseminating straw can be inserted
to this depth without irritation to the vagina. Differences in accessibility of the vagina
could be due to anatomical variations, or could be related to the receptivity of the female
because this behaviour appears to be controlled by the hormonal changes that are
associated with the ovulatory or ovipository cycle [Delville et al. 1986, Shimada and
Saito 1989]. If females could be inseminated at the time when the vagina is the most
accessible, deeper insemination would be possible and more spermatozoa should gain
access to the utero-vaginal storage glands [Brillard et al. 1987].
The longest duration of the fertile period was achieved with inseminations carried
out on the day after oviposition and the declining trend thereafter suggests that a shorter
delay from oviposition to insemination gives a longer fertile period. This relationship
is probably due to the time that the spermatozoa take to reach the storage tubules after
insemination. In the turkey and the chicken, spermatozoa take up to three days to fill
the storage tubules after the artificial insemination [Brillard and Bakst 1990] [Bakst et
al. 1994]. Vaginal contractions associated with oviposition can affect sperm transfer
in the vagina [Brillard 1993], so inseminating female emus on the day of oviposition
could result in less spermatozoa reaching the SSTs and thus a shorter fertile period,
compared with inseminations carried out on other days.
In conclusion, while the insemination dose for the emu is yet to be completely
optimized, we do have a good idea of the number of spermatozoa required for efficient
AI and we can achieve a high male to female ratio in breeding emus, provided they are
inseminated away from the time of oviposition.
Acknowledgement. We thank Miss Caitlin Reed and Mr Peter Cowl for their skilful
technical assistance.
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Ireneusz Artur Malecki, Graeme Bruce Martin
Sztuczne unasienianie emu (Dromaius novaehollandiae) – wpływ
liczby plemników i czasu unasienienia na długość „okresu płodnego”
S t r e s z c z e n i e
Celem badań było określenie długości okresu zapładnialności jaj emu po sztucznym unasienieniu i
wpływu czasu unasienienia na długość tego okresu. Nasienie pobierano za pomocą sztucznej pochwy i
samice unasieniano nierozcieńczonym nasieniem w ciągu 30 minut. Unasieniania dokonywano za pomocą
wziernika, po tym jak samica dobrowolnie usiadła i pozwoliła na dostęp do kloaki. Nasienie deponowano
dopochwowo na głębokość 1-2 cm ze słomki inseminacyjnej osadzonej na strzykawce.
Po jednokrotnym unasienieniu dawką 100, 200 czy 400 milionów plemników samice znosiły
zapłodnione jaja odpowiednio przez okres 10±0,4, 12±0,9 i 15±0,6 dni. Po unasienieniu dawką 400 milionów
plemników w 1, 2 czy 3 dniu cyklu jajowego, długość „okresu płodnego” wyniosła odpowiednio 15,8±1,1,
12,5±2,2 i 10,0±1,5 dni. Wyniki sugerują, że aby uzyskać maksymalną długość okresu zapładnialności jaj
emu w trakcie nieśności, unasienienia powinno się dokonywać w pierwszym dniu cyklu jajowego, to jest
w dzień po zniesieniu jaja.
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